When performing a quantitative analysis of an analysis object, it is normally necessary that the analysis object is measured at two or more respectively different concentration points to form a calibration curve based on its results by a relationship between a signal intensity and a concentration. Depending on the stability of a mass analyzing apparatus, it is sometimes necessary to form the calibration curve every several hours or every day or for every analysis object.
In order to improve the precision of a calibration curve, normally, a calibration curve is formed by using three or more concentration points. This is because the calibration curve sometimes fails to form a straight line for the reasons of saturation of a detector, deviations in measurements and the like.
In the case where a calibration curve is formed for quantifying an analysis object in optical measurements, since the same substance is detected as the same wavelength, it is impossible to measure multiple concentrations at the same time, and the calibration curve needs to be formed by measuring the analysis object at respectively different concentrations. In general, a multi-calibrator capable of calibrating analysis objects of multiple items is used for the biochemical inspections by the optical measurements, but this corresponds to a sample in which analysis objects each at one concentration point that do not interfere with one another are mixed, and this is not a sample including the same substance at multiple concentration points.
On the other hand, also in the mass analyzing apparatus used in the present invention, in order to form a calibration curve for quantifying an analysis object with high precision, it is necessary that the analysis object is measured at least two or more respectively different concentration points to form a calibration curve based on the results of measurement by a relationship between a signal intensity and a concentration.
In a general mass analyzing method, after ionizing an analysis object, various kinds of generated ions are taken in a mass analyzing apparatus, and a measurement intensity for ions is determined for each of the values of mass-to-charge ratio (m/z) that is a ratio of the mass number of ions and the charge thereof. Mass spectrum data obtained as a result include peaks of measurement intensity of the measured ions relative to each of the mass-to-charge ratios. In other words, the mass analyzing apparatus can simultaneously detect the substances as long as the substances have different masses.
Moreover, in an analysis of a sample containing many foreign components as in the case of a biological sample, in an attempt to distinguish an analysis object from its analogue structural molecules such as its metabolites or the like, a tandem mass analyzing method (MS/MS method) is used. In this MS/MS method, among multiple kinds of ions generated from an introduced sample, ions of a specific measurement object component are made to collide with a gas or the like to be dissociated in an analyzing apparatus and the generated ions (product ions) are measured. By using the MS/MS method, analogue structural components can be mutually distinguished with high precision. More specifically, it becomes possible to perform the measurement of only the measurement object in which foreign components that have analogous structure to the measurement object and are not desired to be measured are excluded. Thus, even if there are foreign component ions that have the same mass number as that of the measurement object ions, it is possible to distinguish the measurement object ions when the product ions are different from each other.
When it is desired to accurately quantify an analysis object in a mass analyzing apparatus, in general, a stable isotope compound of the analysis object that is isotope-labeled or a compound that is analogous thereto in chemical and physical properties (hereinafter, referred to as an analogue compound) is used as an internal standard substance. As the internal standard substance, a stable isotope compound and an analogue compound whose response to the mass analyzing apparatus is analogous to the analysis object and which can be measured separately from the analysis object are selected.
In other words, in the measurement in the mass analyzing apparatus, the analysis object, its stable isotope compound and analogue compound exhibit the same behaviors in fluctuations in peak intensity, and in the case where a reduction in the peak intensity, a reduction in ionization efficiency or the like occurs due to any factor such as foreign components, the increase or decrease of a peak area exhibits the same behaviors as that of the analysis object. Herein, when the product ions are detected, the stable isotope compound to be used needs to be a compound in which an element contained in the product ions is isotope-labeled.
More specifically, in order to form a calibration curve for quantifying the analysis object in the mass analyzing apparatus with high precision, it is necessary to prepare two or more solutions obtained by mixing an analysis object and an internal standard substance at different concentration points, and perform the measurement at least twice or more.
As described above, in order to form a calibration curve with high precision, multiple kinds of analysis objects having different concentrations have to be prepared, and the analysis has to be carried out at least twice or more, and consequently, time-consuming tasks are required for the preparations and analysis of those samples. Moreover, there is a possibility that human errors occur when preparing the multiple kinds of quantitative calibrators and performing the measurements thereof.
For this reason, in Japanese Patent Application Laid-Open Publication No. H5-79984 (Patent Document 1), in order to improve the efficiency of an analysis, measurements are performed by using one prepared high-concentration quantitative calibrator while repeating automatic dilution several times as needed, thereby reducing the time-consuming tasks for preparing a plurality of kinds of standard solutions and human errors.
Moreover, Japanese Patent Application Laid-Open Publication No. 2000-65797 (Patent Document 2) has proposed an analyzing method using a stable isotope compound, in which a calibration curve is formed by measuring an analysis object by utilizing a ratio of natural isotopes contained in the analysis object itself.